This grant application is built around previous observations in our laboratory that (i) known inducers of cytochrome P-450, such as 3-methyl cholanthrene (3-MC) and phenobarbital (PB) induce specific forms of mitochondrial cytochrome P-450 which can metabolically activate Benzo(a)pyrene and aflatoxin B1, respectively; and (ii) the two mitochondrial cytochromes P-450, although they represent independent species, show partial homology with microsomal iso-enzymes and are active with both microsomal and mitochondrial type of reductase systems. The objective of this proposal is to purify the 3-MC-induced and PB-induced mitochondrial cytochrome P-450 using a combination of known procedures, including affinity binding to adrenodoxin-sepharose. The purified enzyme will be used for in vitro reconstitution of monooxygenase both in cytochrome P-450 reductase as well as in adrenodoxin + adrenodoxin reductase systems. The heme binding portion of the purified mitochondrial enzymes will be isolated and compared with similar fragments from microsomal enzymes and adrenal mitochondrial P-450scc with respect to antibody cross-reactivity and amino acid composition. Finally, the mRNAs coding for the 3-MC and PB induced forms of mitochondrial cytochromes P-450 will be cloned by the cDNA procedure in E. coli plasmid vectors and the DNA clones will be used for nucleotide sequence analysis to determine the extent of homology with microsomal counterparts. The long-term objective is to study the chromosomal location and organization of genes coding for mitochondrial isoenzymes in relation to microsomal counterparts.